Proc. Natl. Acad. Sci. USA Vol. 85, pp. 985-989, February 1988 Biochemistry Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor ( inhibitor/urokinase/cDNA cloning/bacterial expression) T. M. ANTALIS*t, M. A. CLARK*, T. BARNES*, P. R. LEHRBACH*, P. L. DEVINE*, G. SCHEVZOV*, N. H. Goss*, R. W. STEPHENSt§, AND P. TOLSTOSHEV* *Biotechnology Australia Pty. Ltd., P. 0. Box 20, Roseville, New South Wales 2069, Australia: and tDepartment of Medicine and Clinical Science, Australian National University, Woden Valley Hospital, Garran, Australian Capital Territory 2605, Australia Communicated by Harland G. Wood, October 8, 1987

ABSTRACT Human monocyte-derived plasminogen acti- similar specificity to the placental PAI (7), particularly in the vator inhibitor (mPAI-2) was purified to homogeneity from the presence of fibrin (12). U937 cell line and partially sequenced. Oligonucleotide probes The monocyte-derived PAI (mPAI-2) was originally de- derived from this sequence were used to screen a cDNA library scribed by Golder and Stephens (6) and termed minactivin. prepared from U937 cells. One positive clone was sequenced We have defined this molecule as a PAI-2 type¶ on the basis and contained most of the coding sequence as well as a long of its immunological crossreactivity with the placental PAI. incomplete 3' untranslated region (1112 base pairs). This We designate this molecule mPAI-2. A convenient source of cDNA sequence was shown to encode mPAI-2 by hybrid-select the mPAI-2 is the human histiocytic lymphoma cell line translation. A cDNA clone encoding the remainder of the U937 (7). mPAI-2 mRNA was obtained by primer extension of U937 We report the cloning and sequencing of the cDNA coding poly(A)+ RNA using a probe complementary to the mPAI-2 for mPAI-2 from the U937 cell line11 and its expression in coding region. The coding sequence for mPAI-2 was placed Escherichia coli cells. The deduced sequence under the control of the A PL promoter, and the reveals marked homology to the serine protease inhibitor expressed in Escherichia coli formed a complex with urokinase () superfamily (13) and, in particular, appears to have that could be detected immunologically. By nucleotide se- several striking features in common with the chicken oval- quence analysis, mPAI-2 cDNA encodes a protein containing bumin gene family. 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows MATERIALS AND METHODS extensive homology with members of the serine protease Assays. mPAI-2 activity was measured by a modification inhibitor (serpin) superfamily. mPAI-2 was found to be more (14) of the method of Coleman and Green. Human urokinase homologous to ovalbumin (37%) than the endothelial - was purchased from Calbiochem-Behring. Plasminogen was ogen activator inhibitor, PAI-1 (26%). Like ovalbumin, purified from fresh human plasma by lysine-Sepharose mPAI-2 appears to have no typical amino-terminal signal (Pharmacia) affinity chromatography (15). Endoproteinase sequence. The 3' untranslated region of the mPAI-2 cDNA Lys-C was purchased from Boehringer Mannheim. Affinity- contains a putative regulatory sequence that has been associ- purified to placental inhibitor (16) that crossre- ated with the inflammatory mediators. acted with mPAI-2 were supplied by B. Astedt (University of Lund, Sweden). Plasminogen activators (PA) are serine proteases that medi- Purification of mPAI-2 and Partial Amino Acid Sequence. ate proteolytic cascades involved in cellular translocation, mPAI-2 was purified from the conditioned medium (2 liters) migration, and invasion. These are generally of two types: of U937 cells (American Type Culture Collection CRL 1593) tissue type (tPA), with a high affinity for fibrin and likely the grown in the presence of 4-phorbol 12-myristate 13-acetate major PA in the fibrinolytic system, and urokinase type (PMA) at 30 ng/ml. Concentrated culture supernatants were (uPA), associated with extracellular proteolytic events such applied to a phenyl-Sepharose column equilibrated with 50 as tissue remodeling and destruction, particularly in the mM sodium citrate/2 M NaCI, pH 5.5, and washed with 50 invasive growth and metastatic spread of malignant tumors mM sodium citrate, pH 5.5/0.5 M NaCl/1 mM EDTA, and (reviewed in ref. 1). the mPAI-2 was eluted in 50 mM glycine at pH 9.0. After Regulation of the activity of PA involves specific inhibi- fractionation on Sephacryl S-200, the mPAI-2 preparation tors. These have been classified into four immunologically was electrofocused with a pH gradient of 4.5-6.0. Pure distinct groups: PAT-1 type PA inhibitor from endothelial mPAI-2 was then obtained by reverse-phase (Vydac C4; cells (2, 3); PAI-2 type PA inhibitor from placenta (4, 5), Separations Group, Hesperia, CA) high-pressure liquid monocytes (6), and macrophages (7); the recently described chromatography using a gradient of acetonitrile in 0.1% urinary inhibitor (8); and protease nexin-I (9). The first three PAI are fast acting and specific for PA, whereas protease Abbreviations: PA, plasminogen activator(s); PAI, PA inhibitor(s); nexin-I preferentially inhibits thrombin and has a secondary mPAI-2, monocyte-derived PAI-2; tPA, tissue-type PA; uPA, uro- kinase-type PA; PMA, 4-phorbol 12-myristate 13-acetate. action on plasminogen activation. Endothelial PAI effi- tTo whom reprint requests should be addressed. ciently inhibits tPA and uPA (10), whereas the placental PAI §Present address: Department of Virology, University of Helsinki, is a potent inhibitor of uPA and reacts to a much lesser Haartmaninkatu 3, 00290 Helsinki 29, Finland. extent with tPA (11). The monocyte-derived PAI has a ¶The nomenclature used in this paper is that recommended by the 32nd International Committee on Thrombosis and Hemostasis, June 8, 1986, Jerusalem, Israel. The publication costs of this article were defrayed in part by page charge 1This sequence is being deposited in the EMBL/GenBank data base payment. This article must therefore be hereby marked "advertisement" (Bolt, Beranek, and Newman Laboratories, Cambridge, MA, and in accordance with 18 U.S.C. §1734 solely to indicate this fact. Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03603). 985 986 Biochemistry: Antalis et al. Proc. Natl. Acad. Sci. USA 85 (1988) trifluoroacetic acid (CF3COOH). Following digestion of tants derived from PMA-stimulated U937 cells. The protein mPAI-2 (4 gg) with endoproteinase Lys-C (0.1 Ag) in 20 mM migrated as a single band having a pI of 5-5.2 and Mr of Tris HCI, pH 8.5/5 M urea for 8 hr at 22TC, the resultant 45,000-48,000. It was not possible to derive amino acid peptides were separated by reverse-phase (Snychropak RP- sequence from the intact molecule as the amino terminus of P-C8; SynChrom, Linden, IN) high-pressure liquid chroma- the purified protein was blocked, as has been observed by tography using a gradient of acetonitrile in 0.1% CF3COOH. others (7). Partial amino acid sequences were therefore Partial amino acid sequence was determined by using an determined from selected peptides obtained from an endo- Applied Biosystems (Foster City, CA) 470A gas-phase se- proteinase Lys-C digest of mPAI-2 as described in Materials quencer. The complete details of the purification of mPAI-2 and Methods. These are as follows: peptide will be published elsewhere. 13, Ala-Gln-Ile- Construction of cDNA Libraries. A AgtlO cDNA library was Leu-Glu-Leu-Pro-Tyr-Xaa-Gly-Asp-Val-Xaa-Met-Phe-Leu- constructed with RNA extracted by a modified guanidine HCl Leu-Leu-Pro-Xaa-Glu; peptide 11, Gly-Arg-Ala-Asn-Phe- method (17) from U937 cells that had been cultured in the Ser-Gly-Met-Ser-Glu-Xaa-Asn-Asp-Leu-Phe; peptide 10, presence of PMA at 30 ng/ml for 19 hr. Poly(A) + mRNA was Met-Ala-Glu-Xaa-Glu-Val-Glu-Val-Tyr-Ile-Pro-Gln-Phe- isolated by using oligo(dT)-cellulose (18). Double-stranded Lys-Leu-Glu-Glu-Xaa-Tyr; peptide 6, Leu-Asn-Ile-Gly-Tyr- cDNA was synthesized essentially by the S1 nuclease method Ile-Glu-Asp-Leu-Lys; peptide 9, Ile-Pro-Asn-Leu-Leu-Pro- (19). Following methylation and ligation of EcoRI linkers, Glu-Gly-Xaa-Val. cDNA was fractionated and cDNAs >1000 base pairs (bp) Identification of mPAI-2 Translation Product. In vitro were ligated to phage AgtlO arms, packaged by using a translation of U937 mRNA followed by immunoprecipitation Gigapack (Vector Cloning Systems, San Diego, CA) packag- with anti-placental inhibitor antibodies yielded a translation ing extract, and plated on E. coli C600 lft. The cDNA library product of Mr 43,000-45,000 that was biologically active, as contained -1.5 x 106 independent recombinants. A second evidenced by a shift in mobility in the presence of urokinase library was constructed by 3' extension of U937 mRNA by (Mr 33,000) to Mr 69,000 characteristic of the formation of a using a synthetic oligonucleotide primer derived from the 5' urokinase-mPAI-2 complex (Fig. 1). region of the partially sequenced gene: 5' TTC CAG TAA Isolation of PAI-2 cDNA. The primary cDNA library was ATA ATT CCC TGT GGA TGC ATT 3' (complementary to screened with each of the oligonucleotide probes and cloned nucleotides 391-420; see Fig. 2). Double-stranded cDNA was sequences that hybridized to more than one of the probes synthesized by using the RNase H method (20) and the library were selected for further study. The clone containing the constructed in AgtlO as described above. largest cDNA insert was analyzed by hybrid-select transla- Screening of cDNA Libraries. The sequences of the three tion to verify that the cDNA insert contained sequences that oligonucleotide probes used to screen the primary cDNA encoded mPAI-2. Translation of mRNA that specifically library were predicted by reference to the amino acid se- hybridized to the cDNA insert yielded a product of Mr quence of purified peptide fragments of mPAI-2 and biased 43,000-45,000, consistent with that obtained by immuno- toward human preferred codon usage (21). In the first two precipitation of the mPAI-2 translation product with anti- cases, the sequences were further modified to take into placental inhibitor (Fig. 1). As nucleotide sequence analysis account known highly conserved sequences of other . indicated that the 2100-bp insert did not contain the entire The oligonucleotides were as follows: 5' ATA TGT TTC coding sequence of the mPAI-2 gene, the primer extension CTC GAG CTT GAA CTG AGG GAT GTA CAC CTC GAC approach (25) was used to obtain clones that included the 5' TTC GCT CTC TGC CAT 3', corresponding to peptide 10; terminus of the mRNA. 5' TC ATC AGG CAA CAG GAG GAA CAT GCT CAC cDNA Sequence Analysis. The cDNA sequence of mPAI-2 ATC TCC GGC GTA AGG GAG TTC CAG GAT CTT CAT and the predicted amino acid sequence are shown in Fig. 2. TTT 3', corresponding to peptide 13; and 5' CTC CTC CAG CTT GAA CTG GGG GAT GTA GAC CTC CAC CTC 3', A B corresponding to peptide 10. These probes were labeled by using [y-32P]ATP with T4 polynucleotide kinase (22), and 200- DNA hybridizations were performed under standard condi- tions (23). Final wash stringency was in 0.3 M NaCl/30 mM 0 sodium citrate at 50°C. Phage giving positive signals were 0 92.5- plaque purified and A DNA was prepared as described (22). 0T- -92.5 The library prepared by primer extension was screened by X 69- - using a 29-mer oligonucleotide, 5' GCC TGC AAA ATC -69 GCA TCA GGA TAA CTA CC 3' (complementary to 46- nucleotides 310-335; see Fig. 2), derived from the 5' termi- nus of the partially sequenced gene. Hybridization condi- -46 tions were as above except that the final wash stringency 30- 30-i was 0.15 M NaCl/15 mM sodium citrate at 55°C. DNA Sequence Analysis. cDNA inserts were subcloned -30 into pUC18 for restriction analysis. The DNA sequence was determined by usind the dideoxy chain-termination method 1 2 3 1 2 with subcloned fragments in M13mp9, - 18, and -19 (24). Cell-Free Translations. mRNA was translated in vitro by FIG. 1. Identification of the mPAI-2 translation product follow- using rabbit reticulocyte lysate (Amersham) according to the ing NaDodSO4/polyacrylamide gel electrophoresis. (A) Lane 1, manufacturer's instructions with the addition of 100 ng of total translation products. Lanes 2 and 3, immunoprecipitation of calf liver tRNA per ml. Hybrid-select translations were mPAI-2 translation product by using anti-placental inhibitor anti- carried out according to the method of Maniatis et al. (22). bodies and Pansorbin in the presence (lane 2) and absence (lane 3) of 0.08 international unit of urokinase. (B) Lane 1, pUC18 as control. Lane 2, translation of mRNA selectively hybridized to pBTA447, a RESULTS pUC18 derivative carrying the 2100-bp mPAI-2 cDNA insert. The doublet observed for the in vitro translation product appears to be Purification and Partial Amino Acid Sequence of mPAI-2. the result of a posttranslational modification event that results from mPAI-2 was purified to homogeneity from culture superna- batchwise variation in reticulocyte lysate preparations. Biochemistry: Antalis et al. Proc. Natl. Acad. Sci. USA 85 (1988) 987

1 GTCAGACAGCAACTCAGAGAATAACCAGAGAACAACCAGATTGAAACA 49 79 109 ATG GAG GAT CTT TGT GTG GCA AAC ACA CTC TTT GCC CTC AAT TTA TTC AAG CAT CTG GCA AAA GCA AGC CCC ACC CAG AAC CTC TTC CTC Met Glu Asp Lou Cys Val Ala Asn Thr Lou Ph. Ala Lou Asn Lou Pho Lys His Lou Ala Lys Ala Sor Pro Thr Gln Asn Leu Phe Lou 3C

139 169 199 TCC CCA TGG AGC ATC TCG TCC ACC ATG GCC ATG GTC TAC ATG GGC TCC AGG GGC AGC ACC GAA GAC CAG ATG GCC AAG GTG CTT CAG TTT Ser Pro Trp Sor Io Sor Sor Thr Met Ala Met Val Tyr Mot Gly Sor Arg Gly S*r Thr Glu Asp Gln Met Ala Lys Val Lou Gln Phe 60

229 259 289 AAT GAA GTG GGA GCC AAT GCA GTT ACC CCC ATG ACT CCA GAG AAC TTT ACC AGC TGT GGG TTC ATG CAG CAG ATC CAG AAG GGT AGT TAT Asn Glu Val Gly Ala Asn Ala Val Thr Pro Mot Thr Pro Glu Asn Ph. Thr Sor Cys Gly Pho Mot GIn Gln Il Gln Lys Gly Sor Tyr 90

319 349 379 CCT GAT GCO ATT TTG CAG GCA CAA GCT GCA GAT AAA ATC CAT TCA TCC TTC CGC TCT CTC AGC TCT GCA ATC AAT GCA TCC ACA GGG AAT Pro Asp Ala Ilo Lou Gln Ala Gln Ala Ala Asp Lys Il. His Sor Sor Pho Arg Sor Lou Sor Ser Ala Il Asn Ala Ser Thr Gly Ann 12n 409 439 469 TAT TTA CTG GAA AGT GTC AAT AAG CTG TTT GGT GAG AAG TCT GCG AGC TTC CGG GAA GAA TAT ATT CGA CTC TGT CAG AAA TAT T-C TCC Tyr Lou Lou Glu Sor Val Asn Lys Lou Pho Gly Glu Lys Ser Ala Ser Phe Arg Glu Glu Tyr Ile Arg Leu Cys Gin Lys Tyr Tyr Ser 150

499 529 559 TCA GAA CCC CAG GCA GTA GAC TTC CTA GAA TGT GCA GAA GAA GCT AGA AAA AAG ATT AAT TCC TGG GTC AAG ACT CAA ACC AAA GGC AAA Sor Glu Pro Gln Ala Val Asp Pho Lou Glu Cys Ala Glu Glu Ala Arg Lys Lys Ile Asn Sor Trp Val Lys Thr Gln Thr Lys Gly Lys 180 589 619 649 ATC CCA AAC TTG TTA CCT GAA GGT TCT GTA GAT GGG GAT ACC AGG ATG GTC CTG GTG AAT GCT GTC TAC TTC AAA GGA AAG TGG AAA ACT I1o Pro Asn Lou Lou Pro Glu Gly Sor Val Asp Gly Asp Thr Arg Met Val Lou Val Asn Ala Val Tyr Pho Lys Gly Lys Trp Lys Thr 210 PEPTIDE 9 679 709 739 CCA TTT GAG AAG AAA CTA AAT GGG CTT TAT CCT TTC CGT GTA AAC TCG GCT CAG CGC ACA CCT GTA CAG ATG ATG TAC TTG CGT GAA AAG Pro Pho Glu Lys Lys Lou Asn Gly Lou Tyr Pro Pho Arg Val Asn Ser Ala Gln Arg Thr Pro Val Gln Met Mot Tyr Lou Arg Glu Lys 240

769 799 829 CTA AAC ATT GGA TAC ATA GAA GAC CTA AAG GCT CAG ATT CTA GAA CTC CCA TAT GCT GGA GAT GTT AGC ATG TTC TTG TTG CTT CCA GAT Lou Asn I.e Gly Tyr I1 Glu Asp Lou Lys Al& Gln I.e Lou Glu Lou Pro Tyr Ala Gly Asp Val Sor Met Phe Lou Lou Lou Pro Asp 270 PEPTIDE 6 859 889 919 PEPTIDE 13 GAA ATT GCC GAT GTG TCC ACT GGO TTG GAG CTG CTG GAA AGT GAA ATA ACC TAT GAC AAA CTC AAC AAG TGG ACC AGC AAA GAC AAA ATG Glu Il Ala Asp Vol Sor Thr Gly Lou Glu Lou Lou Glu Sor Glu I1 Thr Tyr Asp Lys Lou Asn Lys Trp Thr Sor Lys Asp Lys Met 300 r 949 979 1009 GCT GAA GAT GAA GTT GAG GTA TAC ATA CCC CAG TTC AAA TTA GAA GAG CAT TAT GAA CTC AGA TCC ATT CTG AGA AGC ATG GGC ATG GAG Ala Glu Asp Glu Val Glu Val Tyr Ile Pro GIn Ph. Lys Lou Glu Glu His Tyr Glu Lou Arg Sor Ile Lou Arg Sor Met Gly Met Glu 330

1 0 3 9 PEPTIDE 10 1 0 6 9 1099 GAC GCC TTC AAC AAG GGA CGG GCC AAT TTC TCA G0G ATG TCG GAG AGG AAT GAC CTG TTT CTT TCT GAA GTG TTC CAC CAA GCC ATG GTG Asp Ala Ph. Asn Lys Gly Arg Ala Asn Ph. Str Gly Mot S*r Glu Arg Asn Asp Lou Phe Lou Sor Glu Val Pho His Gln Ala Met Val 360 r PEPTIDE 11 1 1129 1159 1189 GAT GTG AAT GAG GAG GGC ACT GAA GCA GCC GCT GGC ACA GGA GGT GTT ATG ACA GGG AGA ACT GGA CAT GGA GGC CCA CAG TTT GTG GCA Asp Val Asn Glu Glu Gly Thr Glu Ala Ala Ala GLy Thr Gly Gly Val Mot Thr Gly Arg Thr Gly His Gly Gly Pro Gln Ph. Val Ala 390

1219 1249 1279 GAT CAT CCT TTT CTT TTT CTT ATT ATG CAT AAG ATA ACC AAC TGC ATT *TTA TTT TTC GGC AGA TTT TCC TCA CCC TAA AAC TAA GCG TGC Asp His Pro Ph. Lou Ph. Lou Ile Met His Lys Il Thr Asn Cys I1 Lou Ph. Ph. Gly Arg Ph. Sr Sor Pro *** 420 1309TGCTTC$G.CAAAAGATTTTTGTAGATGAGCTGTGTGCCTCAGAATTGCTATTTCAAATTGCCAAAAATTTAGAGATGTTTTCTACATATTTCTGCTCTTCTGAACAACTTCTGCTACCCA 129C$AAA$AAAAACACAGAAATAATSAGACAATTGTCTATTATAACATGACAACCCTATTAATCATTTGGTCTTCTAAAkATGGGATCATGCCCATTTAGATTTTCCTTACTATCAGTTTATT 1549TTTATAACATTAACTTTTACTTTGTTATTTATTATTTTATATAATGGTGAGTTTTTAAATTATTGCTCACTGCCTATTTAATGTAGCTAATAAAGTTATAGAAGCAGATGATCTGTTAkAT 1669TTCCTAfCTAATAAATGCCTTTAATTGTfTCTCATAATGA~G7AATA^TAGGTATCCCTCCATGCCCTTCTGTAATAAATA~TCTGGAAAAAACATTAAACAATAGGCAAATATATGTTATG gg$9GCATTTCTAGAAATACATAACACATATATATGTCTGTATCTTATATSCAATTGCAAGTATATAATGTCATAATTTCAAGACCAGCCTGGCCAACATAGCGAAACCCTACCTCCACTAAA 1909AATACAGAAATGAGCCGGGAGTGGTGGC6A~aATGGTGAGCACCTGTGATCCCAGCCACTGTGGAGGCCGAGGCAGGACAATCACTTGAACCCAGGAGGCGGAGGCTGCAGTGAGCTGAGA 2029TCGCTCCACTGCACTCCAGCCTGGGCAACAGAGCAAGATTCCATCTCAAAATACATTAAAAAAAAAAACCTATCTGAGGACTCTGAAAAGTAAATGGTAGCAGATAGATTTGAGAAGGGA 2149ACTAGAACTTGAAGCACAATCTATCTGGTGCTCTTTCTTACTTTTGCTTGTTTTCTCCCAATCTTCCAGTCTGGATACAAAGGCAGCCCAATTTCTAGAAATGTATACCAGCCATGAAGA 2269GATAAAGCTCCAAGAGGAGATTTCTCTTTCTGGTATAAGGTATGTGTGTGTATATGGGGGGCGATAAGGTTGGGAGTGTGAGGAATACAGAGTCGGAGAAATCCATTATTTCCACCCTCT 2389CTCTTGCCATTGCAACCAGAC FIG. 2. mPAI-2 cDNA and amino acid sequences. The amino acid residues are numbered beginning with the initiator methionine. The peptides derived from the amino acid sequence of the native mPAI-2 are underlined. The (A + T)-rich sequences in the 3' untranslated region are indicated by dashes.

The translation initiation codon was assigned to the first DISCUSSION in-frame ATG, which is located 49 bp from the 5' end of the cDNA sequence and is preceded by the in-frame stop codon, In the present study, we have cloned the cDNA gene for TAA. The mature mPAI-2, after removal of the initiator mPAI-2 from phorbol ester-induced U937 cells and deduced methionine, is a protein of 415 amino acids of predicted Mr its complete amino acid sequence. In vitro translation of 46,543, which is in good agreement with the molecular mRNA that specifically hybridizes to the cDNA gene yields weight of the unglycosylated form of mPAI-2, as determined a product of Mr 45,000. The product of this cDNA gene by in vitro translation of its mRNA (Fig. 1). It has three expressed in E. coli under the control of the A PL promoter potential N-glycosylation acceptor sites [Asn-Xaa-Thr/Ser is biologically active. (26) at positions 75, 115, and 335] and five cysteine residues. Ye et al. (30) have reported the cloning and expression of Expression in E. coli. The complete coding sequence of a PAI cDNA from human placenta and its expression in E. mPAI-2 was placed under the control of the A PL promoter in coli. Though it was previously known that the placental the vector pLK58 (27) with a synthetic oligonucleotide PAI-2 and the mPAI-2 are immunologically related, compar- inserted upstream of the ATG to provide a bacterial ribo- ison of the placental sequence with the one we have obtained some binding site at an appropriate distance from the start from U937 cells shows that they are very similar. There are codon (28), giving the plasmid pBTA447. Cell extracts of differences between the two sequences in five positions (406, induced E. coli N4830 containing pBTA447 showed biolog- 1227, 1260, 1286, and 1739), resulting in amino acid changes ically active mPAI-2, as evidenced by the shift in electro- in the coding region at three positions, 120, 404, and 413. phoretic mobility in the presence of urokinase, characteristic These are not generally conservative changes, and, in fact, of the formation of a urokinase-mPAI-2 complex (Fig. 3). one results in a serine replaced by a cysteine in the placental 988 Biochemistry: Antalis et al. Proc. Nati. Acad. Sci. USA 85 (1988)

identified in mPAI-2 cDNA. The poly(A) tail and the highly ",PAI 2 MSet Gi, conserved poly(A) addition sequence (31) are missing from GATCIGTGIAAGGAGGTTTAAC ATG GAG... the mPAI-2 sequence. Comparison of the DNA sequences 891 11 o 0 reveals that the mPAI-2 sequence contains an additional 581 0 bp by including a partial Alu repeat (32) of 221 bp (nucleo- v 92- tides 1855-2075), which are not found in the placental cDNA x sequence. 11

L It is likely that both the placental and the mPAI-2s are 68- products of the same gene. The differences observed in the eaoRl amino acid sequences could be the result of polymorphic variation or may be due to sequencing errors. The former z145- would be surprising since the amino acid changes are not conservative in nature. The differences in the 3' region ofthe placental and monocyte cDNAs may result from alternative, 31- perhaps tissue-specific, transcription and splicing events or may represent an anomaly in the cloning procedure. 1231 2 3 Comparison of the predicted amino acid sequence of FIG. 3. Expression of mPAI-2 in E. coli. The DNA fragment mPAI-2 with that of other serine protease inhibitors reveals encoding mPAI-2 (nucleotides 55-1604) and a 26-mer synthetic that mPAI-2 is a member of the serine protease inhibitor oligonucleotide containing a ribosome binding site and the first two superfamily of , known as the serpins (reviewed in codons of mPAI-2 were inserted into the Bgl II site of pLK58 (27) to ref. 34). A sequence alignment of mPAI-2 with five other yield pBTA447. The hatched bar represents the mPAI-2 coding serpins, including the recently described endothelial PAM-i, sequence. This plasmid was then transformed into E. coli N4830 and shows a number of features in common (Fig. 4). Invariant induced by elevating the temperature as described (29). Soluble amino acids are found in 42 positions. Identical amino acids extracts of the cells were partially purified and analyzed by nonre- are found in five of the six serpins, including mPAI-2, in 35 ducing NaDodSO4/polyacrylamide gel electrophoresis. Expression additional positions. The extent of between of mPAI-2 was detected by electrophoretic transfer analysis using homology anti-placental inhibitor antibodies in the absence (lane 3) and in the mPAI-2 and the other five serpins was determined to be 37% presence of M, 33,000 urokinase (lane 1) and M, 55,000 urokinase with chicken ovalbumin, 40% with chicken gene Y, 32% with (lane 2). kb, Kilobases; Apr, ampicillin resistance. III, 28% with a1-antitrypsin, and 26% with sequence reported by Ye et al. (30). The 5' untranslated 1 Following the submission of this manuscript, Webb et al. (33) region of mPAI-2 cDNA is 7 nucleotides shorter than that reported the sequence for human monocyte [Arg]serpin cDNA. The found in the placental PAI-2. The remaining overlapping sequence is identical to that of mPAI-2 within the coding region. sequences in the 5' regions of both clones are identical. A However, the two sequences differ in (i) an additional 22 nucleotides found in the 5' untranslated region of the sequence reported by significant difference is also found in the 3' untranslated Webb et al. (33) and (it) an additional 581-bp 3' untranslated region regions of these two cDNA clones. Following the stop found in mPAI-2 that is not contained in the sequence reported by codon, TAA, a long 3' noncoding region of 1112 bp is Webb et al. (33).

PAI-I M Q M S P A L T C L V L G L A L V F G E G S H H P P S V A H L A M PAI-2 A.V Y OACH ME D[PGC OYCH MIG S I J5 T Al -AT M P S S V S H G I L L L A G L C C L V P V S L A.E D P Q G D A A Q K T D T S H H D Q D H P T F N K I T P N L AT3 M Y S N V I G T V T S G K R K V Y L L S L L L I G F W D C V T CH G S P V D I C T A K P R D I P M N P M C I Y R S P E K K A T E D E G S E Q K I P E A T N R R V W E[JS KA 5D Helix A IlStrand I59 HelixN Helix C PAI-1 SDITJV V XQ [A S K DR G V VLA T M PAI-2 T LIF5LN T-Q N L SPI 9S T M A K[A SIP YFL Is MvfY GS Q MAAI-FD QIFN E EVAGNA AV T P AT PE NF T SC G F MIQS IQ ]S YP D A -I OACH MKI F C F DV1 K[LV H HAA- ElN I JCP II I IA L AM VIY LGIA J~ T Q LN FDIL P------IF GD SSI DYCH AK F H S -T N I FFSP V Al -AT TEA1 Q MSix SIA T A F AMAL L GT LKGLNF L T ------AT3

150 I1J Strand AS I I Helix E IC STRAND Al I I Helix F3 PAI-S J.A-A E LHG~P NK DE I ST'TD-A IF AD ~K G F MPHF F --F R S T VQ V D FS FAFII N JWW~fWTK G M PAI-2 LIQIAQA Kj SS~ S ~ ~ A ST GN VYLL F G A jjM R YIURj IS E VP.lbPA V AVDF LME I NSHWV KT TKG OACH AIQC GT SVNVIH S S~D I LAN TK P N D V ISPFS LA S ILl A 5y PI LPIE L Q V K E L Y G G L EP NF QTA AI A RE L IN SWV E S G DYCH ~~WCGSSEYVHNLFKEL~~~~ELfIFS1E TNP N A T - Y SrL ILYV JF( JV L P[5 LS CARAIR1F YTGVEI K A A NR ~L SIN IHV E K TNG P 1- Al-AT EI EA QIIHIEGIF IEIL ILR T LWQ PD SSQ L QILI TT D G L LC DL L VD KFLE DV K LIIIHFj flA F T 5F IGG-EE AK Q I N fQINGV TQ G AT3 --E K T S[OQ F F LIAN CARL XA N K S SKLLV A [N JDI~]L TffN ~T JQDDII E L VS GA K LfoJP L[ZKEtN QSNA A I NK SN KG + A + +~~~~~~~~~~~~~~~~~~~~~+ AA+ A 20D 250 L Strand A3 I Strand C3 I3 SItrandBl I I Strand PAI-t KGAVDQLTL TPF GS STHRAL N IF T EFTTP PI G DTC VTRLLVNA ALTT R HKjSDGGITV SIV 2AAIQT NWY DGGHIYGYDI LKEL M PAI-2 ~~~~~nLG L NS Q TLRE LIN -IGYIEDLKAn1 ILEL P.L GDj V OACH I R V D ;IQT M V L V N A SI -DL G DYCH I T E D T NR M T 1K S K P V Q PM MC NA N S V A T L P A Al-AT IIVDL K LD T I R V fi1GT E E E GIPHq D Q V T T V PIAA K L G APIN JI SH C K K AT3 NTDVI SEAINEEITVVL TII lSi P NTAKEL Y AGGESCS SAC EGffi1-I-WAVAE-GTM KGD(MI A A + + A 300 Strandl33 1 He1IlixG I I fllixe Strand CS 31 Strand A6 IC Helix I I PAI-1 QJ~~~~~~~~~~~~r~Tg I fSfISH 5NMA T LP L AKPL T M PAI-2 OACH -VS K-ES~~~~~~~~~ DYOtI Al-AT N A I L P D IKIG TH WIITM JLLE N E SA H~P IK L T YGILIK LG Q IG[TIK V lF S IN G- GlV EKA- R G E AT3 T[MVM I L PI PE5K S- AK rE LWP E V[ Q U L L EgMA AML M MP AmE S K Qi D 77JL V mL FSP KK SICPG I V KG + A A + + 350 400 StvandA5 Strand A4 IC Strand Cl StrandNB4 I I StrandAS5 I PAl - 1 M PAr-2 G W MTm T G H G G P Q FF V A D H KI I M OACH DYA-I Al -AT R AT] GD L IV GA I AFFL KE KE MSEKA S A V 121I ALS[-) L N P NRN V T KMN AR EV P T RV A N P C V K

FIG. 4. Homology of mPAI-2 to other serpins. The mPAI sequences were matched by inspection according to the sequence alignment scheme described by Carrell et al. (34), in which the structure is weighted based on the secondary structure of al-antitrypsin. The positions of al-antitrypsin crystal structure according to Loebermann et al. (35) are indicated above the alignment. Amino acids identical to those of mPAI-2 are boxed. The numbers refer to the amino acid residues of mPAI-2. Identity in all six serpins is indicated by an asterisk (*) and in five serpins is indicated by a plus sign (+) (noted beneath the sequences). The sequences of endothelial-type PAI (PAI-1), chicken ovalbumin (OACH), chicken gene Y (DYCH), al-antitrypsin (Al-AT), and antithrombin III (AT3) are from Ginsburg et al. (3), McReynolds et al. (36), Heilig et al. (37), Kurachi et al. (38), and Chandra et al. (39), respectively. Single-letter abbreviations are according to standard usage. Biochemistry: Antalis et al. Proc. Natl. Acad. Sci. USA 85 (1988) 989 PAI-1. The homology of mPAI-2 with ovalbumin and 4. Kawano, T., Morimoto, K. & Uemura, Y. (1970) J. Biochem. chicken gene both of which are members of the serpin (Tokyo) 67, 333-342. Y, 5. Astedt, B., Lecander, I., Brodin, T., Lundblad, A. & Low, K. gene family but have no known inhibitor function, is surpris- (1985) Thromb. Haemostasis 53, 122-125. ingly high, whereas the homology between endothelial PAI-1 6. Golder, J. P. & Stephens, R. W. (1983) Eur. J. Biochem. 136, and mPAI-2 is no more significant than between other 517-522. members of the serpin family. It would appear then that 7. Kruithof, E. K. O., Vassalli, J.-D., Schleuning, W.-D., Mattaliano, mPAI-2 may have diverged from a common ancestor with R. J. & Bachmann, F. (1986) J. Biol. Chem. 261, 11207-11213. ovalbumin and chicken gene Y earlier than PAI-1, in relation 8. Stump, D. C., Thienpont, M. & Collen, D. (1986) J. Biol. Chem. 261, 12759-12766. to other members of the serpin family. A unique difference 9. Scott, R. W. & Baker, J. B. (1983) J. Biol. Chem. 258, 10439-10444. between mPAI-2 and the other serpins is the additional 10. Erickson, L. A., Ginsberg, M. H. & Loskutoff, D. J. (1984) J. Clin. stretch of 32 residues (65-96) in the C-D interhelical region. Invest. 74, 1465-1472. This region is generally either limited to a short 9-residue 11. Saksela, O., Hovi, T. & Vaheri, A. (1985) J. Cell. Physiol. 122, stretch, as in ovalbumin or chicken gene Y, or is absent, as 125-132. 12. Leung, K.-C., Byatt, J. A. & Stephens, R. W. (1987) Thromb. Res. in the other members of the superfamily. The significance of 46, 755-766. this structure is unknown. The active site residues, P1 and 13. Carrell, R. W. & Travis, J. (1985) Trends Biochem. Sci. 10, 20-24. P1', can be assigned to Arg-380 and Thr-381, respectively, in 14. Leung, K.-C., Byatt, J. A. & Stephens, R. W. (1987) Thromb. Res. mPAI-2. As PA converts plasminogen to plasmin by cleav- 46, 767-778. age of an Arg-Val bond (40), this assignment is consistent 15. Deutsch, D. G. & Mertz, E. T. (1970) Science 170, 1095-1096. with the known arginine specificity of PA. 16. Astedt, B., Hagerstrand, I. & Lecander, I. (1986) Thromb. Hae- mostasis 56, 63-65. The amino-terminal sequence of mPAI-2 does not appear 17. Mattick, J. S., Zehner, Z. E. Callabro, M. A. & Wakil, S. J. (1981) to contain a hydrophobic core typical of Eur. J. Biochem. 114, 643-651. sequences (41). Ovalbumin has been shown to contain an 18. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, internal signal sequence (42) corresponding to residues 1408-1412. 253-303 in Fig. 4. The aligned sequence in mPAI-2 is 62% 19. Huynh, T. V., Young, R. A. & Davis, R. W. (1985) in DNA to in this region. This contrasts with Cloning: A Practical Approach, ed. Glover, D. M. (Oxford Univ. homologous ovalbumin Press, Oxford), Vol. 1, pp. 49-78. only 29% homology in this region between ovalbumin and 20. Gubler, U. & Hoffman, B. J. (1983) Gene 25, 263-269. PAI-1, which contains a 23-residue amino-terminal leader 21. Lathe, R. (1985) J. Mol. Biol. 183, 1-12. sequence (3). Unfortunately, blockage of the amino terminus 22. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular of mature native mPAI-2 precludes direct amino acid analy- Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, sis of the amino-terminal sequence. Cold Spring Harbor, NY). region of 23. Amersham International (1985) Membrane Transfer and Detection The partial Alu repeat in the 3' untranslated Methods (Amersham International, Amersham, England). mPAI-2 is preceded by several short stretches (10-20 bp) of 24. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. (A+ T)-rich sequences (nucleotides in regions 1543-1593 and Sci. USA 74, 5463-5467. 1791-1862) that are similar to those found in several transiently 25. Luse, D. S., Haynes, J. R., Van Leeuwen, D., Schon, E. A., expressed genes leading to mRNA instability (43). It also Cleary, M. L., Shapiro, S. G., Lingrel, J. B. & Roeder, R. G. (1981) includes the consensus sequence, TTA'1T'TAT, described by Nucleic Acids Res. 9, 4339-4354. 26. Bause, E. (1983) Biochem J. 209, 331-336. Caput et al. (44) as being common to a number of inflammatory 27. Botterman, J. & Zabeau, M. (1985) Gene 37, 229-239. mediators, including tumor necrosis factor, the interferons, and 28. Tessier, L.-H., Sondermeyer, P., Faure, T., Dreyer, D., Bena- the interleukins. Caput et al. (44) have proposed that these vente, A., Villeval, D., Courtney, M. & Lecocq, J.-P. (1984) sequences are involved in the control of gene expression, at Nucleic Acids Res. 12, 7663-7675. either a transcriptional or translational level. 29. Mott, J. E., Grant, R. A., Ho, Y.-S. & Platt, T. (1985) Proc. Natl. 88-92. of mPAI-2 may be to neutralize PA Acad. Sci. USA 82, The physiological role 30. Ye, R. D., Wun, T.-C. & Sadler, J. E. (1987) J. Biol. Chem. 262, at extravascular sites during tissue remodeling or metastatic 3718-3725. growth of malignant tumors, but little is currently known 31. Proudfoot, N. J. & Brownlee, G. G. (1976) Nature (London) 263, about the regulation of mPAI-2 expression, the relevance and 211-214. significance of its close relationship to ovalbumin, or the role 32. Jelinek, W. R. & Schmid, C. W. (1982) Annu. Rev. Biochem. 51, of glycosylation in the secretion and stability of this protein. 813-844. 33. Webb, A. C., Collins, K. L., Snyder, S. E., Alexander, S. J., The availability of biologically active mPAI-2 produced from Rosenwassen, L. J., Eddy, R. L. Shows, T. B. & Auron, P. E. E. coli will allow detailed investigations of the biological (1987) J. Exp. Med. 166, 77-94. function of mPAI-2 in the inhibition of plasminogen activation 34. Carrell, R. W. & Boswell, D. R. (1986) in Proteinase Inhibitors, ed. and its role in extracellular proteolytic processes. Barrett, A. & Salvesen, G. (Elsevier/North-Holland Biomedical, Amsterdam), pp. 403-420. We thank Ian D. Walker for the amino acid sequence analyses, 35. Loebermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R. (1984) Neil Willetts for helpful discussions and critical reading of the J. Mol. Biol. 177, 531-556. manuscript, Kin-chuen Leung for helpful discussions, Bernie Mc- 36. McReynolds, L., O'Malley, B. W., Nisbett, A. D., Fothergill, J. oligonucleotides, Jan Maitland, Gillian E., Givol, D., Fields, S., Robertson, M. & Brownlee, G. G. (1978) Inerney for synthesis of Nature (London) 273, 723-728. Morgan, Ken Bertram, and Roslyn McIntyre for excellent cell 37. Heilig, R., Muraskowsky, R., Kloepfer, C. & Mandel, J. L. (1982) culture and technical assistance, and Rhonda Smith for typing the Nucleic Acids Res. 10, 4363-4382. manuscript. This work was supported in part by funds from the 38. Kurachi, K., Chandra, T., Degen, S. J. F., White, T. T., Mar- National Biotechnology Program Grants Scheme of Australia, chioro, T. L., Woo, S. L. C. & Davie, E. W. (1981) Proc. Natl. which is administered by the Commonwealth Department of Indus- Acad. Sci. USA 78, 6826-6830. try, Technology and Commerce. 39. Chandra, J., Stackhouse, R., Kidd, V. J. & Woo, S. L. C. (1983) Proc. Natl. Acad. Sci. USA 80, 1845-1848. 1. Dano, K., Andreason, P. A., Grondahl-Hansen, J., Kristensen, P., 40. Collen, D. (1980) Thromb. Haemostasis 43, 77-89. Nielson, L. S. & Skriver, L. (1985) Adv. Cancer Res. 44, 139-266. 41. Perlman, D. & Halvorsen, H. 0. (1983) J. Mol. Biol. 167, 391-409. 2. Loskutoff, D. J., Van Mourik, J. A., Erickson, L. A. & Lawrence, 42. Lingappa, V. R., Lingappa, J. R. & Blobel, G. (1979) Nature D. (1985) Proc. Natl. Acad. Sci. USA 80, 2956-2960. (London) 281, 117-121. 3. Ginsburg, D., Zeheb, R., Yang, A. Y., Rafferty, U. M., Andrea- 43. Shaw, G. & Kamen, R. (1966) Cell 46, 659-667. son, P. A., Nielsen, L., Dano, K., Lebo, R. V. & Gelehrter, T. 44. Caput, D., Beutler, B., Hartog, K., Thayer, R., Brown-Shimer, S. D(1986) J. Clin. Invest. 78, 1673-1680. & Cerami, A. (1986) Proc. NatI. Acad. Sci. USA 83, 1670-1674.